This application discloses improved methods for isolating SCM-responding lymphocytes for the performance of the SCM test useful in the detection of cancer and other diseases and conditions.
Many diseases occurring in humans and animals can be detected by the presence of foreign substances, particularly in the blood, which are specifically associated with a disease or condition. Tests for antigens or other such substances produced as a result of such diseases show great promise as a diagnostic tool for the early detection and treatment of the particular disease that produced the antigen or other substance. Procedures for the detection of such substances must be reliable, reproducible, and sensitive in order to constitute a practical diagnostic procedure for health care providers. In addition any such procedure should be able to be carried out by persons of ordinary skill and training in laboratory procedure, and should be relatively fast and inexpensive.
For example, in the treatment of the various malignancies that afflict humans and animals, referred to generally as cancer, it is recognized that early detection is a key to effective treatment, especially as many therapeutic procedures are effective only in relatively early stages of the disease. In fact, virtually all known cancer treatments are not only more effective, but safer, when administered in early stages of cancer. Far too many cases of cancer are only discovered too late for effective treatment.
Accordingly, there is a great need for rapid, easy-to-perform, and reliable tests which can diagnose cancer at early stages. In this connection, new tests and procedures are being developed to effect early diagnosis of the cancer.
We have developed and reported one such test for the early detection of cancer in L. Cercek, B. Cercek, and C. I. V. Franklin, "Biophysical Differentiation Between Lymphocytes from Healthy Donors, Patients with Malignant Disease and Other Disorders," Brit. J. Cancer 29, 345-352 (1974) and L. Cercek and B. Cercek, "Application of the Phenomenon of Changes in the Structuredness of Cytoplasmic Matrix (SCM) in the Diagnosis of Malignant Disorders: a Review", Europ. J. Cancer 13, 903-915 (1977), which are incorporated herein by this reference.
Our basic SCM test includes the steps of:
(1) challenging a selected subpopulation of lymphocytes from a donor with a challenging agent such as a mitogen or an antigen associated with a condition or disease, such as cancer; and
(2) determining the change in structuredness of the cytoplasmic matrix SCM) of the challenged lymphocytes, typically using fluorescence polarization.
SCM reflects the forces of interaction between macromolecules of the cell and other cellular components such as water molecules, ions, adenosine triphosphate, and cyclic adenosine phosphate. Perturbations of these interactions result in changes in SCM.
When applied to cancer, our SCM test is based on the phenomenon that the internal structure of a selected subpopulation of the lymphocytes from a healthy individual is altered when challenged by a mitogen such as phytohaemagglutinin, but is not altered by other selected challenging agents, such as cancer basic protein (CaBP) and/or antigens derived from specific malignant tumors such as tumor associated antigens (TAAs). Contrarily, the equivalent subpopulation of lymphocytes from an individual with cancer responds oppositely. In other words the same subpopulation of lymphocytes from cancer patients does not respond in the SCM test when challenged by a mitogen, but does respond when challenged by a number of cancer-associated antigens. When TAAs derived from specific malignant tumors are used as challenging agents, only lymphocytes from individuals with the same type of tumor from which the TAA had been isolated will respond in the SCM test.
The changes seen in SCM are believed to reflect changes in the internal structure of the lymphocyte as the lymphocyte is activated for synthesis Similar changes can occur in living cells other than lymphocytes during the cell cycle and growth of the cells. Such changes can also be evoked by various external agents, such as ionizing radiation, mechanical forces, chemicals, growth inhibiting and stimulating agents, etc. These changes can be conveniently monitored with a specially adapted technique of fluorescein fluorescence polarization, as we have published in numerous articles, including L. Cercek and B. Cercek, "Studies on the Structuredness of Cytoplasm and Rates of Enzymatic Hydrolysis in Growing Yeast Cells. I. Changes Induced by Ionizing Radiation," Int. J. Radiat. Biol. 21, 445-453 (1972); L. Cercek and B. Cercek, "Studies on the Structuredness of Cytoplasm and Rates of Enzymatic Hydrolysis in Growing Yeast Cells. II. Changes Induced by Ultra-Violet Light," Int. J. Radiat. Biol. 22, 539-544 (1972); L. Cercek and B. Cercek, "Relationship Between Changes in the Structuredness of Cytoplasm and Rate Constants for the Hydrolysis of FDA in Saccharomyces cerevisiae," Biophysik 9, 109-112 (1973); L. Cercek, B. Cercek, and C. H. Ockey, "Structuredness of the Cytoplasmic Matrix and Michaelis-Menten Constants for the Hydrolysis of FDA During the Cell Cycle in Chinese Hamster Ovary Cells," Biophysik 10, 187-194 (1973); B. I. Lord, L. Cercek, B. Cercek, G. P. Shah, T. M. Dexter and L. G. Lajtha, "Inhibitors of Haemopoietic Cell Proliferation: Specificity of Action Within the Haemopoietic System," Brit. J. Cancer 29, 168-175 (1974); L. Cercek and B. Cercek, "Involvement of Cyclic-AMP in Changes of the Structuredness of Cytoplasmic Matrix," Radiat. & Environ. Biophys. 11, 209-212 (1974); L. Cercek, P. Milenkovic, B. Cercek, & L. G. Lajtha, "Induction of PHA Response in Mouse Bone Marrow Cells by Thymic Extracts as Studied by Changes in the Structuredness of Cytoplasmic Matrix," Immunology 29, 885.gtoreq.891 (1975); L. Cercek and B. Cercek, "Effects of Osmomolarity, Calcium and Magnesium Ions on the Structuredness of Cytoplasmic Matrix SCM)," Radiat. & Environ. Biophys. 13, 9-12 (1976); L. Cercek & B. Cercek, "Changes in the Structuredness of Cytoplasmic Matrix (SCM) Induced in Mixed Lymphocyte Reactions," Radiat & Environ Biophys. 13, 71-74 (1976); L. Cercek, B. Cercek, & C. H. Ockey, "Fluorescein Excitation and Emission Polarization Spectra in Living Cells: Changes During the Cell Cycle," Biophys J. 23, 395-405 (1978); L. Cercek and B. Cercek, "Effect of Osmolality and Density of Gradients on the Isolation of SCM-Responding Lymphocytes," Brit. J. Cancer 38, 163-165 (1978); L. Cercek and B. Cercek, "Involvement of Mitochondria in Changes of Fluorescein Excitation and Emission Polarization Spectra in Living Cells," Biophys J. 28, 403-412 (1979); L. Cercek, B. Cercek, and B. I. Lord, "The Effect of Specific Growth Inhibitors on Fluorescein Fluorescence Polarization Spectra in Haemopoietic Cells," Brit. J. Cancer. 44, 749-752 (1981 ); and L. Cercek and B. Cercek, "Effects of Ascorbate Ions on Intracellular Fluorescein Emission Polarization Spectra in Cancer and Normal Proliferating Cells," Cancer Detection and Prevention 10, 1-20 (1987), all of which are incorporated herein by this reference.
The usefulness of this SCM test for the detection of cancer has been documented in numerous articles. Articles from our laboratory include; L. Cercek, B. Cercek, and J. V. Garrett, "Biophysical Differentiation Between Normal Human and Chronic Lymphocytic Leukaemia Lymphocytes," in Lymphocyte Recognition and Effector Mechanisms (K. Lindahl-Kiessling and D. Osoba eds., New York, Academic Press, 1974), pp. 553-558; L. Cercek, B. Cercek and C. I. V. Franklin, "Biophysical Differentiation between Lymphocytes from Healthy Donors, Patients with Malignant Disease and Other Disorders," Brit. J. Cancer 29, 345-352 (1974); L. Cercek and B. Cercek, "Changes in the SCM Response Ratio (RR.sub.SCM)) After Surgical Removal of Malignant Tissue," Brit. J. Cancer 31, 250-251 (1975); L. Cercek and B. Cercek, "Apparent Tumour Specificity with the SCM Test," Brit. J. Cancer 31, 252-253 (1975); L. Cercek and B. Cercek, "Changes in the Structuredness of Cytoplasmic Matrix of Lymphocytes as a Diagnostic and Prognostic Test for Cancer," in Cell Biology and Tumour Immunology, Excerpta Medica International Congress Series No. 349, Proceedings of the XI International Cancer Congress, Florence, 1974 (Amsterdam, Excerpta Medica, 1974), vol. 1, pp. 318-323; L. Cercek and B. Cercek, "Application of the Phenomenon of Changes in the Structuredness of Cytoplasmic Matrix (SCM) in the Diagnosis of Malignant Disorders: a Review," Europ. J. Cancer 13, 903-915 (1977); L. Cercek and B. Cercek, "Detection of Malignant Diseases by Changes in the Structuredness of Cytoplasmic Matrix of Lymphocytes Induced by Phytohaemagglutinin and Cancer Basic Proteins," in Tumour Markers, Determination and Clinical Role: Proceedings of the Sixth Tenovus Workshop, Cardiff, April 1977 (K. Griffith, A. M. Neville, and C. G. Pierrepoint, eds., Cardiff, Alpha Omega Publishing Co., (1978), pp. 215-226) and L. Cercek and B. Cercek, "Changes in SCM-Responses of Lymphocytes in Mice After Implantation with Ehrlich Ascites Cells," Europ. J. Cancer 17, 167-171 (1981), all of which are incorporated herein by this reference.
The usefulness of the SCM test has been confirmed in articles from other laboratories, including F. Takaku, K. Yamanaka, and Y. Hashimoto, "Usefulness of the SCM Test in the Diagnosis of Gastric Cancer," Brit. J. Cancer 36, 810-813 (1977); H. Kreutzmann, T. M. Fliedner, H. J. Galla, and E. Sackmann, "Fluorescence-Polarization Changes in Mononuclear Blood Leucocytes After PHA Incubation: Differences in Cells from Patients with and Without Neoplasia," Brit. J. Cancer 37, 797-805 (1978); Y. Hashimoto, T Yamanaka, and F. Takaku, "Differentiation Between Patients with Malignant Diseases and Non-Malignant Diseases or Healthy Donors by Changes of Fluorescence Polarization in the Cytoplasm of Circulating Lymphocytes," Gann 69, 145-149 (1978); J. A. V. Pritchard and W. H. Sutherland, "Lymphocyte Response to Antigen Stimulation as Measured by Fluorescence Polarization (SCM-Test)," Brit. J. Cancer 38 339-343 (1978); J. A. V. Pritchard, J. E. Seaman, I. H. Evans, K. W. James, W. H. Sutherland, T. J. Deeley, I. J. Kerby, I. C. M. Patterson, and B. H. Davies, "Cancer-Specific Density Changes in Lymphocytes Following Stimulation with Phytohaemagglutinin," Lancet 11, 1275-1277 (Dec. 16, 1978); H. Orjasaeter, G. Jordfald, and I. Svendsen, "Response of T-Lymphocytes to Phytohaemagglutinin (PHA) and to Cancer-Tissue-Associated Antigens, Measured by the Intracellular Fluorescence Polarization Technique (SCM Test)," Brit. J. Cancer 40, 628-633 (1979); N. D. Schnuda, "Evaluation of Fluorescence Polarization of Human Blood Lymphocytes (SCM Test) in the Diagnosis of Cancer," Cancer 46, 1164-1173 (1980); J. A. V. Pritchard, W. H. Sutherland, J. E. Siddall, A. J. Bater, I. J. Kerby, T. J. Deeley, G. Griffith, R. Sinclair, B. H. Davies, A. Rimmer, & D. J. T. Webster, "A Clinical Assessment of Fluorescence Polarisation Changes in Lymphocytes Stimulated by Phytohaemagglutinin (PHA) in Malignant and Benign Disease," Europ. J. Cancer, Clin. Oncol. 18,651-659 (1982); G. R. Hocking, J. M. Rolland, R. C. Nairn, E. Pihl, A. M. Cuthbertson, E. S. R Hughes, and W. R. Johnson, "Lymphocyte Fluorescence Polarization Changes After Phytohaemagglutinin Stimulation in the Diagnosis of Colorectal Carcinoma," J. National Cancer Inst. 68, 579-583 (1982); M. Deutsch and A. Weinreb, "Validation of the SCM-Test for the Diagnosis of Cancer," Eur. J. Cancer, Clin. Oncol. 19, 187-193 (1983); S. Chaitchik, O. Asher, M. Deutsch, and A. Weinreb, "Tumour Specificity of the SCM Test for Cancer Diagnosis," Europ. J. Cancer, Clin. Oncol. 21, 1165-1170 (1985); and J. Matsumoto, T. Tenzaki and T. Ishiguro, "Clinical Evaluation of Fluorescein Polarization of Peripheral Lymphocytes (SCM Test) in the Diagnosis of Cancer," J. Japan Soc. Cancer Ther. 20, 728-734 (1985), all of which are incorporated herein by this reference.
As reported, the SCM test indicates usefulness both as a screening test for the general detection of malignancies and as a test to diagnose specific types of malignancies. This has been confirmed by blind clinical tests and corroborated by other investigators.
The SCM test can be applied to detection of diseases and conditions other than cancer, such as viral and bacterial infections, including the detection of the AIDS virus, determination of allergic reactions, tissue typing, and monitoring of allograft rejections such as organ transplant rejection based on the SCM responses in mixed lymphocyte reactions, as disclosed in the 1976 Radiation and Environmental Biophysics article by L. Cercek and B. Cercek. The presence of other antigen-producing diseases and bodily conditions does not interfere with the SCM test; a patient afflicted with more than one type of antigen-producing disease can be tested for a multiplicity of such diseases simply by running separate tests using for each test an antigen derived from each separate disease or condition being tested for.
When fluorescence polarization is used to determine changes of SCM, such changes are seen as a decrease in the fluorescence polarization of the cells when polarized light is used to excite an intrinsic fluor generated intracellularly by the hydrolysis of a non-fluorescent compound which has been absorbed by the lymphocytes. The fluor typically is fluorescein and the non-fluorogenic compound is typically fluorescein diacetate (FDA). The FDA serves as a fluorogenic agent precursor. An extrinsic fluor is used because the intrinsic fluorescence of cellular components is too small to give meaningful results in this test. Therefore, all references to fluorescence polarization values herein are references to fluorescence polarization values obtained with an extrinsic fluor, preferably one generated by enzymatic hydrolysis from a non-fluorogenic compound added to and absorbed by the cells. Typically, excitation of the fluor occurs with light at 470 nm and the intracellular fluorescein fluorescence polarization peak occurs at 510 nm. Excitation at 442 nm can also be used, in which case the intracellular fluorescein polarization peak occurs at 527 nm.
Intracellular fluorescence polarization is a measure of resistance to rotational relaxation of fluorescein molecules; the greater the intracellular resistance to rotation, the greater the measured fluorescence polarization. As seen in the SCM test, a decrease in fluorescence polarization is believed to result mainly from changes in the conformation of the mitochondria, the energy-producing organelles of the cell. Changes in the mitochondria are believed to result from the contractions of the cristae or inner folds of the mitochondrial membrane, as stated in L. Cercek & B. Cercek, "Involvement of Mitochondria in Changes of Fluorescein Excitation and Emission Polarization Spectra in Living Cells," Biophys. J., 28, 403-412 (1979).
Immunologically the SCM-responding lymphocytes are T-cell mononuclear leukocytes Although not fully understood, it is believed that SCM-responding lymphocytes are involved in the recognition of antigens that are circulating in the blood stream. This recognition of antigens triggers the body's immune system. Accordingly, these cells become primed to recognize foreign substances, such as antigens, produced by the disease or condition affecting the body. Not all lymphocytes found in the bloodstream, however, are SCM-responding; only selected sub-populations of cells having specific and narrowly defined buoyant densities are SCM-responding.
In order to carry out the SCM test successfully, preferably the selected sub-population of SCM-responding lymphocytes must be isolated from other blood components, including all non-SCM-responding lymphocytes. In our prior work, as described in the 1977 European Journal of Cancer article, we isolated SCM-responding lymphocytes on Ficoll.TM.-Triosil.TM. gradients after removal of the phagocytic cells from the blood sample by treatment with iron powder or carbonyl-iron powder. The SCM-responding lymphocytes isolated were those that floated on top of a Ficoll-Triosil gradient with a density of 1.081 g/cm.sup.3 and an osmolality of 0.320 Osm/kg, while non-responding cells banded inside the gradient. The density figure of 1.081 g/cm.sup.3 refers to the density of the gradient solution itself; the actual buoyant density of the SCM-responding cells themselves was not determined, but must be somewhat less than 1.081 g/cm.sup.3. The SCM-responding cells were collected from the gradient with a Pasteur pipette, taking great care to avoid the collection of blood components separated above the narrow band of SCM-responding lymphocytes or cells that separated in the density gradient solution below the narrow band. This procedure requires considerable practice and manual dexterity to carry out properly. Additionally, the conditions for isolation and separation of the SCM-responding lymphocytes, such as the temperature of the blood sample and the density gradient solution, and the density, osmolality, and pH of the solution must be rigorously controlled. For example, the temperature should be controlled to .+-.0.2.degree. C. Failure to control any of these isolation and separation conditions can result either in loss of some of the desired SCM-responding cells or the isolation of some of the non-responding cells along with the desired SCM-responding cells, as stated in L. Cercek & B. Cercek, "Effect of Osmolality and Density of Gradients on the isolation of SCM-Responding Lymphocytes," Brit. J. Cancer 38, 163-165 (1978). Such errors can result in loss of sensitivity of the SCM test. Indeed, a number of other laboratories attempting to corroborate our work have failed to obtain reliable and reproducible results because of poor or unskilled technique in the separation of the SCM-responding lymphocytes.
This need for precise separation of the SCM-responding lymphocytes became even more important when it was realized that there exists more than one subpopulation of SCM-responding cells, and that these subpopulations yield markedly different responses in the SCM test. The existence of such subpopulations was reported in the 1978 Lancet article by Pritchard et al., in the 1982 European Journal of Cancer Clinical Oncology article by Pritchard et al., and in J. M. Rolland, R. C. Nairn, A. P. Nind, and E. Pihl, "Significance of Lymphocyte Fluorescence Polarization Changes After Phytohaemagglutinin Stimulation in Cancer and Non-cancer Conditions," J. National Cancer Institute 72, 267-273 (1984). These studies revealed that besides the subset of SCM-responding cells that had been isolated in previous SCM studies, there exists an additional subset of SCM-responding cells with a buoyant density different from that of the first set. Unlike the SCM-responding cells previously studied, this additional subset of cells only responded to PHA, and did so only when isolated from cancer patients. It did not respond to any cancer-associated antigens such as CaBP, whether or not isolated from patients with malignancies, and failed to respond to PHA when isolated from patients without cancer. In summary, the response of this subset of cells to PHA is exactly the opposite of that of the first subset. Clearly, it is necessary to separate these two subsets of cells in performing the SCM assay. Otherwise, the results are impossible to interpret, especially when PHA is used to stimulate the SCM-responding cells.
The techniques used by Pritchard et al. and Rolland et al. to separate the two types of SCM-responding cells from each other and from other blood components were slight modifications of the technique described by us in our 1977 European Journal of Cancer article. In this technique, the two fractions of cells are isolated in rather ill-defined bands or zones near the interface between the blood plasma and the Ficoll-Triosil density gradient. In the use of such a technique, it has proven difficult to avoid cross-contamination of the cell fractions with each other and with SCM non-responding lymphocytes. Such cross-contamination is apparent, for example, in the results shown in Table 1 of the 1982 European Journal of Cancer Clinical Oncology article by Pritchard et al., where many patients show responses to PHA in both fractions of SCM-responding cells. Since only one fraction or the other of the SCM-responding cells actually responds to PHA, depending on whether or not the cells are isolated from patients with cancer, such a dual response clearly indicates the presence of cross-contamination.
An additional disadvantage of the prior methods of lymphocyte isolation has been the use of iron powder or carbonyl-iron powder in an initial step of removing the phagocytic cells from the blood sample. The use of such materials can cause toxic effects to the lymphocytes or their hemolysis. These deleterious effects, presumably caused by impurities in some batches of the iron powder or carbonyl-iron powder, occur sporadically, but are difficult to control when they do occur.
Accordingly there is a need for a method of isolating SCM-responding lymphocytes that can be carried out rapidly by workers with limited training, that does not require special dexterity, and that can reliably separate both subpopulations of SCM-responding cells without cross-contamination or contamination of either of the sub-populations with SCM-non-responding cells Preferably such a technique can also dispense with the use of carbonyl-iron powder or iron powder in the initial step of the procedure.